Biomedical Engineering Reference
In-Depth Information
that can be removed (
stuffer
) and replaced by foreign
DNA (
replacement
vectors). Since phage
can accom-
modate only about 5% more than its normal com-
plement of DNA, vector derivatives are constructed
with deletions to increase the space within the
genome. The shortest
λ
P
L
P
R
t
L
t
R1
cl
N
t
R2
O
P
red
DNA molecules that produce
plaques of nearly normal size are 25% deleted.
Apparently, if too much non-essential DNA is
deleted from the genome, it cannot be packaged into
phage particles efficiently. This can be turned to
advantage for, if the replaceable fragment of a
replacement-type vector is either removed by phys-
ical separation or effectively destroyed by treatment
with a second restriction endonuclease that cuts it
alone, then the deleted vector genome can give rise
to plaques only if a new DNA segment is inserted into
it. This amounts to positive selection for recombin-
ant phage carrying foreign DNA.
Many vector derivatives of both the insertional and
replacement types were produced by several groups
of researchers early in the development of recombin-
ant DNA technology (e.g. Thomas
et al.
1974, Murray
& Murray 1975, Blattner
et al.
1977, Leder
et al.
1977). Most of these vectors were constructed for
use with
Eco
RI,
Bam
HI or
Hin
dIII, but their applica-
tion could be extended to other endonucleases by
the use of linker molecules. These early vectors have
been largely superseded by improved vectors for
rapid and efficient genomic or complementary DNA
(cDNA) library construction (see Chapter 6).
λ
int
Q
P
′
R
S
R
cos
J
A
F
Z
Legend
Early transcripts
Middle transcripts
Late transcripts
Fig. 4.12
Major promoters and transcriptional termination
sites of phage
λ
. (See text for details.)
and patterns of expression therefore overlap. The
cro
product, when sufficient has accumulated, prevents
transcription from P
L
and P
R
. The gene
Q
is ex-
pressed from the distal portion of the extended P
R
transcript and is responsible for the middle-to-late
switch. This also operates by anti-termination. The
Q
product specifically anti-terminates the short P
R
transcript, extending it into the late genes, across
the cohered
cos
region, so that many mature phage
particles are ultimately produced.
Both
N
and
Q
play positive regulatory roles essen-
tial for phage growth and plaque formation; but an
N
−
phage can produce a small plaque if the termina-
tion site t
R2
is removed by a small deletion termed
nin
(
N
-independent) as in
Improved phage-
λ
vectors
As with plasmid vectors, improved phage-vector
derivatives have been developed. There have been
several aims, among which are the following.
• To increase the capacity for foreign DNA frag-
ments, preferably for fragments generated by any
one of several restriction enzymes (reviewed by
Murray 1983).
• To devise methods for positively selecting recom-
binant formation.
• To allow RNA probes to be conveniently prepared
by transcription of the foreign DNA insert; this
facilitates the screening of libraries in chromosome
walking procedures. An example of a vector with
this property is
N
−
nin
.
λ
Vector DNA
Wild-type λ DNA contains several target sites for
most of the commonly used restriction endonu-
cleases and so is not itself suitable as a vector.
Derivatives of the wild-type phage have therefore
been produced that either have a single target site
at which foreign DNA can be inserted (
insertional
vectors) or have a pair of sites defining a fragment
ZAP (see p. 93).
• To develop vectors for the insertion of eukaryotic
cDNA (p. 93) such that expression of the cDNA, in
λ
